Bacteriocin from lactococcus lactis

ABSTRACT

A bacteriocin produced by Lactococcus lactis NRRL-B-18535 is described. The bacteriocin is useful in foods and other materials and has a wide spectrum of activity against Gram-positive bacteria in a pH range between 2 and 8.

This is a divisional of application Ser. No. 07/492,969 filed on Mar.13, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel bacteriocin derived from aLactococcus and method of use to inhibit bacteria, particularly in foodsand other materials in need of protection from the bacteria. The presentinvention particularly relates to a bacteriocin produced by Lactococcuslactis LL-1 deposited as NRRL-B-18535 (previously known as Streptococcuslactis).

2. Prior Art

The lactic streptococci have been previously described to produce avariety of polypeptide antibiotics, diplococcin, lactostrepcins andbacteriocins (Klaenhammer, T. R., Biochemie 70: 337-349 (1988)). Theterm nisin describes a family of polypeptide antibiotics produced byLactococcus lactis that prevents the outgrowth of Clostridium andBacillus spores (Eapen, K. C., et al., J. Food. Sci. Technol. 20:231-240 (1983)). Bacteriocins are also produced by pediococci.

Diplococcin is an antimicrobial agent produced by Lactococcus cremoris.This inhibitor does not inhibit sporeformers and is only active againstother dairy lactococci (Davey, G. P. and B. C. Richardson., Appl.Environ. Microbiol. 41:84-89 (1981)).

Lactostrepcins are inhibitory proteins produced by the lactococci thatinhibit other streptococci. These molecules are active at relatively lowpH and activity is completely lost when the pH is raised to 7.0 (Kozak,W., et al., J. Dairy Res. 45: 247-257 (1978)).

Bacteriocins produced by lactic lactococci have been observed in manycommercial strains (Geis, A., et al., Appl. Environ. Microbiol.45:205-211 (1983)). Eight bacteriocin types (I-VIII) have beenidentified on the basis of their activity spectrum, proteolytic enzymesusceptibility, heat stability and cross-reaction with other bacteriocinproducers (Geis, A., et al. Appl. Environ. Microbiol. 45:205-211(1983)).

The problem is that the bacteriocins are not active over a wide pHrange. It would be very desirable to provide a bacteriocin which isuseful in a wide variety of foods regardless of whether they are acidicor basic.

OBJECTS

It is therefore an object of the present invention to provide a novelbacteriocin which is effective at a pH between pH 2 and 8. It is furtheran object of the present invention to provide a bacteriocin which can berelatively easily isolated from a particular strain of Lactococcuslactis. These and other objects will become increasingly apparent byreference to the following description and the drawings.

IN THE DRAWINGS

FIG. 1 is a high pressure liquid chromatographic (HPLC) amino acidprofile of the bacteriocin of the present invention.

GENERAL DESCRIPTION

The present invention relates to a bacteriocin produced by a Lactococcuswhich comprises: a protein having a molecular weight of about 6000daltons, which is inactivated by protease and not inactivated byalpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits thegrowth of bacteria selected from the group consisting of Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcuspentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcuslactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus,Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillusplantarum and has an optimal pH for inhibition between about pH 2 and 8.

Further the present invention relates to a method for inhibitingGram-positive bacteria which can occur with a material which comprises:providing a bacteriocin with the material in an effective amount whichinhibits the Gram-positive bacteria, wherein the bacteriocin is derivedfrom a Lactococcus lactis and wherein the bacteriocin is a proteinhaving a molecular weight of about 6000 daltons, is inactivated byprotease and not inactivated by alpha-chymotrypsin, trypsin, lipase,pepsin and lysozyme, inhibits the growth of bacteria selected from thegroup consisting of Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcusacidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostocmesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum,Lactobacillus bifermentans and Lactobacillus plantarum and has anoptimal pH for inhibition between about pH 2 and 8.

Further the present invention relates to a composition which comprises:an unspoiled food system which is spoiled by Gram-positive bacteria anda bacteriocin derived from cells of a Lactococcus lactis, wherein thecomposition contains an amount of the bacteriocin to provide betweenabout 10 and 100,000 AU of the bacteriocin per gram of the food systemsufficient for the bacteriocin to inhibit the Gram-positive bacteria andwherein the bacteriocin is a protein having a molecular weight of about6000 daltons, is inactivated by protease and not inactivated byalpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibits thegrowth of bacteria selected from the group consisting of Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcuspentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcuslactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus,Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillusplantarum and has an optimal pH for inhibition between about pH 2 and 8.

Further the present invention relates to a device which comprises: amaterial on the device which can become infected with Gram-positivebacteria, and a bacteriocin provided with the material in an amountsufficient to inhibit the Gram-positive bacteria, wherein thebacteriocin is from cells of a Lactococcus lactis and is a proteinhaving a molecular weight of about 6000 daltons, is inactivated byprotease and not inactivated by alpha-chymotrypsin, trypsin, lipase,pepsin and lysozyme, inhibits the growth of Staphylococcus aureus,Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcuspentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcuslactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus,Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillusplantarum and has an optimal pH for inhibition between about pH 2 and 8.

Further the present invention relates to a method for producing abacteriocin which comprises: incubating live cells of a Lactococcuslactis in a growth medium for the cells so as to produce the bacteriocinin the growth medium, and wherein the bacteriocin is a protein having amolecular weight of about 6000 daltons, is inactivated by protease andnot inactivated by alpha-chymotrypsin, trypsin, lipase, pepsin andlysozyme, inhibits the growth of bacteria selected from the groupconsisting of Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus carnosus, Pediococcus pentosaceus, Pediococcusacidilactici, Lactococcus cremoris, Lactococcus lactis, Leuconostocmesenteroides, Lactobacillus bulgaricus, Lactobacillus fermentum,Lactobacillus bifermentans and Lactobacillus plantarum and has anoptimal pH for inhibition between about pH 2 and 8.

The strain Lactococcus lactis LLA 1.0 has been deposited under theBudapest Treaty with the Northern Regional Research Laboratory inPeoria, Ill. as NRRL-B-18535. It is available only upon request by nameand deposit number. The strain has the following fermentationcharacteristics: it is able to ferment dextrose, mannitol, sucrose,maltose, salicin, rhamnose, trehalose, cellobiose, mannose, fructose andN-acetyl-glucosamine.

The strain had three resident plasmids measuring about 33.42, 28.57 and5.82 Mdal in size.

SPECIFIC DESCRIPTION

The following Examples show the production of the bacteriocin fromLactococcus lactis NRRL-B-18535 and its use in foods and othermaterials. It also shows the amino acid profile of a hydrolyzate of thebacteriocin. The determination of the approximate molecular weight isalso shown.

EXAMPLE 1 Production of the Bacteriocin

Bacterial Strains and Media

The bacterial strains used in this study were routinely grown on MRSlactobacillus broth (Difco, Detroit, Mich.).

Bacteriocin Assay

Production of bacteriocin was assayed by spotting cells on MRS agar(Difco Laboratories, Detroit, Mich.) that contained 2.0% MES (N-morpholino! ethanesulfonic acid buffer; Sigma, St. Louis, Mo.). Theseplates were incubated at 35° C. for 18 hours. Assay plates were exposedto chloroform vapor for 30 minutes and overlaid with soft agar (0.075%)seeded with indicator cells. Plates were incubated at 32° C. for 18hours. Isolates producing a clear zone were considered as producingbacteriocin.

Inhibitory Spectrum of LL-1

The plate assay system was used to evaluate the spectrum of bacteriocinactivity. The strain NRRL-B-18535 showed activity against strains ofStaphylococcus aureus, S. epidermidis, S. carnosus, Pediococcuspentosaceus, P. acidilactici, Lactococcus cremoris, Lactococcus lactis,Lactobacillus fermentum, Lactobacillus bifermentans, Leuconostocmesenteroides, Lactobacillus bulgaricus and L. plantarum. Strains ofStreptococcus mutans, S. sanguis, S. faecalis and Listeria monocytogeneswere not sensitive to the bacteriocin LL-1. The strain was resistant tonisin.

EXAMPLE 2 Purification and Characterization of the Bacteriocin

One liter of MRS broth (Difco) was inoculated at 1% with an 8 hour oldculture of LLA 1.0 grown in the medium of Example 1 and was incubatedstatically at 32° C. for 24 hours. After 24 hours the cells were removedby centrifugation at 16,000×g for 20 minutes at 4° C. The supernatantwas filtered through a 0.22 micron (pore size) filter (Millipore Corp.,Bedford, Mass.). The supernatant was assayed for bacteriocin activity byspotting 5 microliters of a serial two-fold dilution series onto MRSplates overlaid with soft agar seeded with indicator cells. Assay plateswere incubated at 35° C. The indicator strain was Pediococcuspentosaceus FBB63C. One arbitrary unit (AU) of bacteriocin was definedas 5 microliters of the highest dilution of culture supernatant yieldinga definite zone of growth inhibition on the indicator lawn. The titerwas expressed as the reciprocal of the highest dilution showinginhibition.

Ammonium sulfate (Sigma Chemical Co., St. Louis, Mo.) was added to thefiltered supernatant to 50% (wt/vol) saturation at 4° C. Afterprecipitation for 18 hours at 4° C., the mixture was centrifuged at16,000×g for 15 minutes at 4° C. The precipitate was reconstituted in 25ml of 0.05M sodium citrate buffer, pH 6.0. The reconstituted precipitatewas dialyzed against the 0.05M sodium citrate buffer at 4° C. by usingSpectra/Por no. 6 membrane tubing (Spectrum Medical Industries, Inc.,Los Angeles, Calif.) and the titer of its activity was determined. Thereconstituted dialyzed precipitate was then subjected to furtherpurification with gel filtration chromatographs using Spectra/Gel AcA202(Spectrum Medical Industries, Inc., Los Angeles, Calif.).

The bacteriocin preparation (6 ml) was applied to an ascendingSpectra/Gel AcA 202 column (2.6 by 30 cm) in 0.05M sodium citrate buffer(pH 6.0). Fractions were collected (4 ml) and assayed for bacteriocinactivity. The active factors were then collected and concentrated10-fold in the dialysis tubing by the removal of water with Carbowax 20(Fisher Scientific Co., Pittsburgh, Pa.). This active concentratedfraction was then applied to an ascending Spectra/Gel AcA 202 column(1.6 by 60 cm) in 0.05M sodium citrate buffer (pH 6.0). The titer of thepartially purified bacteriocin was determined and was used for partialcharacterization of the bacteriocin.

Effects of Heat Treatment and Enzymes

A partially purified sample of bacteriocin LL-1 (6,400 AU/ml) wasassayed for thermostability and enzymatic effects on activity. Thebacteriocin was incubated with each enzyme at a final concentration of50 micrograms/ml for 60 minutes. Incubation in the presence ofalpha-chymotrypsin and trypsin was at 25° C., and all otherenzyme-bacteriocin mixtures were incubated at 37° C. Inactivation of theenzymes was achieved by boiling them for 3 minutes. It is considered tobe a type VI bacteriocin because the strain that produces it isresistant to nisin and does not inhibit S. sanguis. Temperaturestability of the bacteriocin was assessed by heating a solution ofbacteriocin to 80° C. for 60 minutes, 100° C. for 10 minutes, and 121°C. for 15 minutes. After each treatment, bacteriocin samples wereassayed for activity.

Enzymes

All enzymes were obtained from Sigma. Alpha-chymotrypsin (type II; 47U/mg) and lipase (type 1; 8.6 U/mg) were dissolved in 0.05M Trishydrochloride (pH 8.0) containing 0.01M CaCl₂ ; protease (type V; 1U/mg), lysozyme (grade I, 41, 400 U/mg) and trypsin (type IX; 15,000U/mg) were dissolved in 0.05M Tris hydrochloride (pH 8.0); and pepsin(3,200 U/mg) was dissolved in 0.2M citrate buffer (pH 6.0).

pH Stability of Activity

Partially purified bacteriocin (1 ml) was dialyzed against buffers ofvarious pH's. The bacteriocin solution (12,800 AU/ml) was dialyzed for18 hours with 2 changes against 0.05M glycine hydrochloride buffer (pH2.0), 0.05M citrate buffer (pH 3 to 6), 0.05M Tris hydrochloride (pH 7to 9), and 0.05M carbonate-bicarbonate buffer (pH 10 to 11). Afterdialysis, the contents of the tubing were assayed for bacteriocinactivity. The bacteriocin LL-1 was sensitive to protease and notsensitive to alpha-chymotrypsin, trypsin, lipase, pepsin or lysozyme.The bacteriocin was observed to be most stable from pH 2-8, with someloss in activity at pH 9 and 10. Approximately one-fourth of theactivity was still present at pH 11.0. Exposure of the bacteriocin to121° C. did destroy all of the LL-1 activity. Boiling at 100° C. for 10minutes resulted in 75% loss in activity of the bacteriocin. This 75%activity loss was also observed at 80° C. for 60 minutes.

EXAMPLE 3 Nutritional Studies

Each of the media listed in Table 1 was prepared in 100 ml quantities.

The media were adjusted to pH 6.8 before autoclaving. The media wereinoculated with an 8 hour culture of NRRL-B-18535 at a rate of 1% andthen incubated at 32° C. for 24 hours. After 24 hours, 25 ml of theabove culture was centrifuged at 24,000×g for 15 minutes at 4° C. Thesupernatant was then filter sterilized using a 0.22 micron filter(Millipore, Bedford, Mass.) and tested for the least titer whichinhibited Pediococcus pentosaceus FBB63C as the indicator strain.

The results of the nutritional study are depicted in Table 1.

                  TABLE 1    ______________________________________    Nutritional Studies of NRRL-B-18535 for the    Production of Bacteriocin LL-1.    Media                    Titer    ______________________________________    Brain Heart Infusion Broth (Difco, Detroit, MI)                             0    Tryptic Soy Broth (Difco, Detroit, MI)                             0    All Purpose Tween Broth (Difco, Detroit, MI)                               1:2+    MRS Lactobacillus Broth (Difco, Detroit, MI)                             1:8    MRS + 1% yeast extract (Oxoid, Basingstoke,                               1:4+    England)    MRS + 1% Hy-Soy ™ (Sheffield Products,                             1:8    Norwich, NY)    MRS + 1% Hy-Case ™ (Sheffield Products,                             1:8    Norwich, NY)    10% peptonized milk with 1% glucose                             0    10% peptonized whey with 1% glucose                             0    Corn Steep Base (I)    4% cornsteep    3% yeast extract    5% glucose with:    (a)      Nothing               1+    (b)      1% N-Z Amine Type AS ™                                   1+             (Sheffield Products, Norwich, NY)    (c)      1% PRIMATONE SGM ™                                   1+             (Sheffield Products, Norwich, NY)    (d)      1% EDAMIN K ™    1             (Sheffield Products, Norwich, NY)    (e)      1% PRIMAGEN P ™  1:2             (Sheffield Products, Norwich, NY)    (f)      1% PRIMATONE HS ™                                 1:2             (Sheffield Products, Norwich, NY)    (g)      1% EDAMIN S ™      1+             (Sheffield Products, Norwich, NY)    (h)      1% AMICASE T ™   1:2             (Sheffield Products, Norwich, NY)    (i)      1% AMICASE ™     1:2             (Sheffield Products, Norwich, NY)    (j)      1% PRIMATONE ™   1:2             (Sheffield Products, Norwich, NY)    (k)      1% AMISOY ™        1:2+             (Sheffield Products, Norwich, NY)    (l)      1% HYSOY TYPE T ™                                 1             (Sheffield Products, Norwich, NY)    (m)      1% PRIMATONE G ™ 1:2             (Sheffield Products, Norwich, NY)    (n)      1% PRIMAGEN ™      1:2+             (Sheffield Products, Norwich, NY)    CORN STEEP BASE (II)    4% cornsteep    5% dextrose, with:    (a)      2% AMISOY ™      1:2    (b)      2% PRIMAGEN ™    ______________________________________

The most effective medium for the production of bacteriocin LL-1 appearsto be MRS broth that is unsupplemented. Other media were not aseffective and protein hydrolysate supplements did not stimulatebacteriocin production. Whey or milk based media were the leasteffective for the production of LL-1.

EXAMPLE 4 Production of Dried Bacteriocin LL-1

Lactococcus lactis NRRL-B-18535 was grown in one liter of MRS broth(Difco, Detroit, Mich.) for 24 hours at 32° C. The cells were pelletedby centrifugation at 16,000×g at 4° C., and the supernatant wascollected. The supernatant was then filter sterilized with a 0.22 micronpore size filter. Nonfat dry milk powder was added to 10%(weight/volume) to facilitate drying. This mixture was lyophilized intoa dry powder.

EXAMPLE 5 Minimum Inhibitory Concentration (MIC) of the LyophilizedBacteriocin LL-1 Against Pediococcus pentosaceus FBB63C

The bacteriocin LL-1 powder of Example 4 was dissolved in APT broth orTryptic Soy Broth and two-fold serially diluted to concentrationsranging from 1000 AU/ml to 2.0 AU/ml. Approximately 1×10³ Pediococcuspentosaceus/ml were added to each of the tubes which were then incubatedfor 24 hours at 35° C. The MIC value was the lowest concentration tubedisplaying no visible turbidity. The results are summarized in Table 2.

                  TABLE 2    ______________________________________    Strain            MIC    ______________________________________    Pediococcus pentosaceus                      8.0 AU/ml    ______________________________________

EXAMPLE 6 Minimum inhibitory concentration (MIC) of the lyophilizdbactriocin LL-1 against Straphylococcus aureus 265

The bacteriocin LL-1 powder of Example 4 was dissolved in APT broth orTryptic Soy Broth and two-fold serially diluted to concentrationsranging from 1000 AU/ml to 2.0 AU/ml. Approximately 1×10³ Staphylococcusaureus/ml were added to each of the tubes which were then incubated for24 hours at 35° C. The MIC value was the lowest concentration tubedisplaying no visible turbidity. The results are summarized in Table 3.

                  TABLE 3    ______________________________________    Strain           MIC    ______________________________________    Staphylococcus aureus                      1000 AU/ml    ______________________________________

EXAMPLE 7 Molecular Weight Determination of the Bacteriocin LL-1

The molecular weight of the bacteriocin of Example 1 was determined bygel filtration. 1.5 ml (800 AU/ml) was applied to an ascendingSpectra/Gel AcA 202 column (1.6 by 60 cm: Spectrum, Los Angeles, Calif.)in 0.05M sodium citrate buffer (pH 6.0). The elution volume of thebacteriocin was compared to the elution volumes of standard proteins.Bacteriocin activity was determined as described above. The proteinstandards and their molecular weights included the following: cytochromeC, 12,400; aprotinin, 6,500; melittin, 2,846 (Sigma).

The bacteriocin preparations were examined on 12% SDS-PAGE gel. Samplesand molecular weight standards 1 mg/ml were dissolved in sample bufferand loaded on the gel. The material was then subjected toelectrophoresis for 1 hour at 16 mA and then for 1 hour at 24 mA. Thegel was then stained with silver strain (BioRad, Richmond, Calif.) orassayed for bcteriocin activity by a direct detection system (Bhunia, A.K. et al, Appl. Bacteriol. 65:261-268 (1988)).

The molecular weight was observed to be approximately 6,000 daltons fromgel filtration. The SDS gel overlay with Pediococcus pentosaceusconfirmed the approximate size observed with gel filtration.

EXAMPLE 8 Amino Acid Profile of Purified Bacteriocin LL-1

The bacteriocin was purified as previously described in Example 7. Anactive concentrated fraction from the Spectra/Gel AcA202 column wassubjected to further purification using a C-8 analytical column.Fractions were assayed for bacteriocin activity. The active fractionswere then further concentrated using the Speed Vac Concentrator™ (SavantInstruments Inc. Farmingdale, N.Y.) and resuspended in 50 microliters ofdistilled water. This material was then analyzed for amino acid contentusing a modification of the PICO-TAG™ system (Waters Associates,Milford, Mass.). The method involves sample hydrolysis followed byderivatization with phenylisothiocyanate and subsequent analysis by HPLCMundt, M. O., W. G. Beattie, and F. R. Wieland, J. Bacteriol. 98:938-942(1969)!. Amino acids were identified by comparing the retention times ofa known standard to that of the active fraction hydrolyzate (FIG. 1.)The results listed in Table 4 compare the ratios of the various aminoacids observed to glutamic acid, which was observed in the greatestamount.

                  TABLE 4    ______________________________________    Ratios of various amino acids to glutamic acid.    Amino Acid    Ratio to glutamic acid    ______________________________________    Aspartic acid 0.42    Glutamic acid 1.0    Serine        0.35    Glycine       0.83    Histidine     0.16    Arginine      0.35    Threonine     0.34    Alanine       0.76    Proline       0.48    Tyrosine      0.19    Valine        0.43    Methionine    0.14    Cysteine      0.12    Isoleucine    0.27    Leucine       0.64    Phenylalanine 0.19    Lysine        0.50    ______________________________________

EXAMPLE 9 Salad Dressing With Added Contaminant Microorganisms

The following Tables 5, 6 and 7 show the use of the bacteriocin LL-1 insalad dressing to which Lactobacillus fermentum NRRL-B-18586 has beenadded. The spoilage bacterium strain is one which is very active in foodspoilage.

                  TABLE 5    ______________________________________            Desig-         Spoilage        Dilution            nation                  Dressing.sup.1                           Bacteria.sup.2                                     LL-1.sup.3                                           Buffer.sup.4    ______________________________________    Uninoculated              A       100 g    0       0     3.9 ml    Control    Inoculated              B       100 g    0.1 ml (10.sup.6)                                       0     3.9 ml    Control    Inoculated              C       100 g    0.1 ml (10.sup.6)                                       0.78 ml                                             3.12 ml    plus    bacteriocin    LL-1 (200 Au/g)    Inoculated              D       100 g    0.1 ml (10.sup.6)                                       3.9 ml                                             0    plus    bacteriocin    LL-1    (1000 AU/g)    ______________________________________     .sup.1 Dressing used was Marie's ™ Ranch Dressing. The general     composition of the salad dressing was soybean oil, fresh buttermilk, whol     eggs, egg yolks, distilled vinegar, sugar, salt, spices, garlic, onion an     xanthan gum.     .sup.2 Lactobacillus fermentum NRRLB-18586 streptomycin resistant,     addition about 3.0 × 10.sup.3 cfu/g of salad dressing.     .sup.3 Bacteriocin LL1 used was a 50% ammonium sulfate preparation that     was dialyzed against 0.05M sodium citrate buffer (pH 6.0), then filter     sterilized. (Concentration: 25,600 AU/ml). The experiment was conducted a     25° C. for ten days.     .sup.4 The dilution buffer was used to provide equivalent volumes of adde     fluid.

                  TABLE 6    ______________________________________    Bacterial counts in the Salad Dressing.            Time (days)    Designation              T-0.sup.a    T-1      T-10    ______________________________________    A         <10.sup.2b   <10.sup.2                                    <10.sup.2    B         4.0 × 10.sup.3                           3.1 × 10.sup.3                                    7.0 × 10.sup.2    C         3.0 × 10.sup.3                           2.2 × 10.sup.3                                    2.5 × 10.sup.2    D         2.3 × 10.sup.3                           3.5 × 10.sup.2                                    <10.sup.2    ______________________________________     .sup.a Time (days)     .sup.b Bacterial Counts (cfu/g) Lactobacillus fermentum 18586 Sm.sup.r

                  TABLE 7    ______________________________________    Organoleptic Evaluation of Salad Dressing.    Designation   T-10.sup.a    ______________________________________    A             Not spoiled    B             Spoiled.sup.b    C             Not spoiled    D             Not spoiled    ______________________________________     .sup.a Time (days)     .sup.b Acetic acid was detected by smell and taste, and gas (CO.sub.2) wa     produced and observed in the dressing as evidence of spoilage.

This Example shows that the bacteriocin LL-1 was effective to inhibitspoilage bacteria introduced into a salad dressing.

EXAMPLE 10 Salad Dressing With Natural Contaminant Microorganisms

The following Tables 8 and 9 demonstrate the inhibition of the normal,lactic acid spoilage flora using the bacteriocin LL-1 in salad dressing:

                  TABLE 8    ______________________________________                                        Dilution             Designation                     Dressing.sup.1                               LL-1.sup.2                                        Buffer    ______________________________________    Uninoculated               A         100 g     0      3.9 ml    Control    Uninoculated               B         100 g     0.78 ml                                          3.12 ml    plus bacteriocin    LL-1 (200 AU/g)    Uninoculated plus               C         100 g     3.9 ml 0    bacteriocin    LL-1 (1000 AU/g)    ______________________________________     .sup.1 Dressing used was Marie's ™ Ranch Dressing. The general     composition of the salad dressing was soybean oil, fresh buttermilk, whol     eggs, egg yolks, distilled vinegar, sugar, salt, spices, garlic, onion an     xanthan gum. Because of the natural ingredients there is a high level of     lactic bacteria such as Lactobacillus fermentum NRRLB-18586 in the salad     dressing.     .sup.2 Bacteriocin LL1 used was a 50% ammonium sulfate preparation that     was dialyzed against 0.05M sodium citrate buffer (pH 6.0), then filter     sterilized. (Concentration: 25,600 AU/ml). The dilution buffer was used t     provide equivalent volumes of added liquid with LL1.     The experiment was conducted at 25° C. for ten days.

                  TABLE 9    ______________________________________    Bacterial counts in the salad Dressing.              Time (days)    Designation T-0.sup.a    T-2    T-10    ______________________________________    A           <10.sup.2b   <10.sup.3                                    10.sup.8    B           10.sup.2     <10.sup.2                                    10.sup.5    C           10.sup.2     <10.sup.2                                    10.sup.3    ______________________________________     .sup.a Time (days)     .sup.b Bacterial Counts (cfu/g) Lactobacillus sp. spoilage agents

The results show that LL-1 was very effective in inhibiting the growthof the lactic bacteria naturally present in the salad dressing.

The bacteriocin LL-1 was stable in various environments. The bacteriocinwas effective in reducing the initial contaminant bacteria load andmaintaining this protection over a period of several days. The sameresults can be achieved in various food systems such as gravies, meats,vegetables and the like, which can be raw or cooked, and particularlycoleslaw, macaroni salad, potato salad and sausages. The bacteriocin isparticularly effective where raw foods are mixed with other ingredientswhich promote the growth of Gram-positive bacteria naturally present onfood. The bacteriocin can be effective on such items as bandages,sanitary napkins and ointments (liquids and powders) used for woundhealing. In general the bacteriocin can also be useful in the form ofointments (liquids, or powders) as disinfectant for animate andinanimate objects where Staphylococcus aureus is a problem. Thebacteriocin can also be used to treat wounds in mammals which can beinfected with Gram-positive bacteria.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

We claim:
 1. A purified bacteriocin produced by a Lactococcus whichcomprises:a protein having a molecular weight of about 6000 daltons,which is inactivated by protease V and not inactivated byalpha-chymotrypsin, trypsin, lipase, pepsin and lysozyme, inhibitsgrowth of bacteria selected from the group consisting of Staphylococcusaureus, Staphylococcus epidermidis, Staphylococcus carnosus, Pediococcuspentosaceus, Pediococcus acidilactici, Lactococcus cremoris, Lactococcuslactis, Leuconostoc mesenteroides, Lactobacillus bulgaricus,Lactobacillus fermentum, Lactobacillus bifermentans and Lactobacillusplantarum and has a pH for inhibition between about pH 2 and 8 whereinthe purified bacteriocin has a high pressure liquid chromatographicamino acid profile as shown in FIG. 1 as a result of hydrolysis of thebacteriocin to produce the amino acids and labeling of the amino acidswith phenyl isothiocyanate, wherein the ratio of amino acids to glutamicacid are as follows:

    ______________________________________    Amino Acid Ratio to glutamic acid    ______________________________________           Aspartic acid                    0.42           Glutamic acid                    1.0           Serine   0.35           Glycine  0.83           Histidine                    0.16           Arginine 0.35           Threonine                    0.34           Alanine  0.76           Proline  0.48           Tyrosine 0.19           Valine   0.43           Methionine                    0.14           Cysteine 0.12           Isoleucine                    0.27           Leucine  0.64           Phenylalanine                    0.19           Lysine   0.50    ______________________________________     wherein the bacteriocin is produced by Lactococcus lactis NRRLB-18535 in     culture medium.

wherein the bacteriocin is produced by Lactococcus lactis NRRL-B-18535in a culture medium.